The control of proliferation of animal cells during normal development and the loss of control in the conversion of normal cells to cancer cells is of central importance in human growth, aging, and disease. In order for cells to divide they must first replicate their DNA, and the control of initiation of DNA replication is closely coupled to the control of cell division in eukaryotic systems. Understanding the molecular mechanisms of DNA replication in eukaryotic cells is fundamental to gaining an understanding of those control systems. To gain a better understanding of the process of DNA replication in eukaryotic cells, we propose to continue a study of the replication apparatus of the yeast Saccharomyces cerevisiae. The main objects of the study are small circular DNA plasmids which replicate in yeast under nuclear control. Both acentric ring plasmids derived exclusively from yeast replication origins cloned in Escherichia coli bacterial vectors will be used. Additional modified plasmids will be constructed and the effects of the modifications on the replication of the plasmids in yeast will be compared with unmodified plasmids. The mechanism of amplification of multi-copy yeast plasmids to high copy-number will be investigated. Replicative intermediates of modified and unmodified acentric plasmids will analyzed to investigate steps in the initiation mechanism. Yeast mutations isolated which influence DNA replication will be mapped using molecular of a cell-free DNA replication system for the yeast plasmids. The genetics including recombinant DNA technology, DNA analysis by several types of gel electrophoresis, DNA sequence analysis, yeast and bacterial genetics using molecular and classical methods, and biochemical fractionation and analysis of components involved in DNA replication. This will provide them with motivation and research experience to prepare them for a biomedical career.